In recent years, proteins, polypeptides, synthetic compounds, compounds extracted from natural resources and the like having a physiological activity have been found in a large number, and the application thereof to pharmaceutical products has been actively studied. However, such physiologically active substances show a short serum half-life when administered in vivo, and a sufficient pharmacological effect is often difficult to achieve. This is because, in general, a physiologically active substance administered in vivo mostly disappears from the body due to glomerular filtration in the kidney, macrophage uptake in the liver or spleen, and the like. Accordingly, attempts have been made to improve in vivo behavior by including such physiologically active substance in a liposome or polymer micelle, increasing the molecular weight by chemically modifying polyethylene glycol, which is an amphiphilic polymer, and the like. Polyethylene glycol shows low interaction with other biogenic substances due to its steric repulsion effect and the hydrated layer it has. As a result, a polypeptide (e.g., protein, enzyme etc.) modified with polyethylene glycol can avoid biological reactions such as glomerular filtration in the kidney, immunoreaction and the like when administered to the body, thus affording a longer serum half-life than non-modified ones. In addition, modification with polyethylene glycol affords the effect of lowering the toxicity and antigenicity of the modified substance, and further enhances the solubility of a highly hydrophobic, slightly water-soluble compound.
Conventionally, when a physiologically active substance is to be modified with polyethylene glycol, particularly when a low molecular weight pharmaceutical agent or peptide is to be modified, only a few kinds of reactive functional groups can be used for modification with polyethylene glycol. Moreover, when a number of polyethylene glycol molecules are used for the modification to ensure a sufficient effect of modification with polyethylene glycol, problem occurs in that the active sites of peptide or pharmaceutical agent, which is the substance to be modified, are blocked, thus preventing sufficient expression of the function and efficacy possessed by the substances themselves to be modified, or sufficient water solubility cannot be afforded.
To solve such problems, attempts have been made to use a branched polyethylene glycol derivative that reduces the sites modified by polyethylene glycol. JP-B-61-42558 proposes polyethylene glycol-modified L-asparaginase, and discloses a derivative wherein two polyethylene glycols as reactive polyethylene glycol are bonded to cyanuric chloride. However, cyanuric chloride, which is a starting material of the reactive polyethylene glycol derivative, has three reactive sites having equivalent reactivity. Thus, it is difficult to synthesize highly pure polyethylene glycol-modified L-asparaginase by selectively introducing thereinto two different polyethylene glycol chains.
Moreover, U.S. Pat. Nos. 5,643,575 and 5,932,462 propose branched polyalkylene glycol derivatives, suggesting a derivative having two or more polyalkylene glycol chains. Specifically, however, only a two-branch type having two polyalkylene glycol chains is disclosed. Furthermore, WO02/060978 proposes a branched derivative having three or more polyalkylene glycol chains. However, the polyalkylene glycol chain used does not have a terminal functional group. Specifically, only a compound having one kind of functional group, wherein the same polyalkylene glycol chains are bonded to equivalent groups, is disclosed. From the aspect of production process, it is difficult to introduce various polyalkylene glycol chains and many kinds of functional groups into the compound.